Despite our considerable knowledge of ras protein structure and biochemistry, we remain ignorant both of the precise biological function of normal ras proteins in normal cell growth and of the mechanism whereby oncogenic ras proteins trigger the aberrant growth of malignant cells. The recent observation that the posttranslational modification of ras proteins by a farnesyl isoprenoid (an essential intermediate in cholesterol biosynthesis) is critical for ras membrane association and transforming activity has prompted two new directions for ras studies: 1) specifically blocking the farnesyl modification of ras proteins as a novel method of rational drug design for cancer treatment, and 2) determining the significance of this lipid modification for normal and oncogenic ras biological activities. The experimental studies in this proposal are based on the recent observations that the substitution of the farnesyl isoprenoid on normal ras by a geranylgeranyl isoprenoid gave rise to a new class of dominant inhibitory ras mutant protein, whereas the substitution of the fatty acid myristate gave rise to a transforming protein. Either of these lipids can promote the membrane association and biological function of oncogenic ras proteins. Therefore, although membrane association is critical for ras function, normal and oncogenic ras proteins apparently possess different requirements for this association that may reflect their regulation of distinctly different mitogenic signal transduction pathways. The specific aims of this proposal are to determine (1) the role of protein prenylation in the biological activity of normal and oncogenic ras proteins, (2) the role of plasma membrane association in ras biological activity and whether this role differs for normal and oncogenic proteins, (3) to determine the basis for specific modification of proteins by specific types of lipids (isoprenoids versus fatty acids), and (4) whether the particular properties of lipid modification and subcellular location of a potential new oncogene protein, TC21, are shared with ras. These aims will be approached by comparing the biochemistry and biology of authentically farnesylated ras to mutant forms of ras that modified by heterologous lipids. These studies should contribute significantly to understanding the biochemical function of normal and oncogenic ras proteins and provide an important foundation for understanding the contribution of different lipid modifications to the function of other proteins, including src, the heterotrimeric G proteins, and ras-related proteins, that regulate diverse normal cellular processes.